382 The Immunoassay Handbook
FIGURE 1 Example of immunometric format ELISA.
383CHAPTER 5.1 Practical Guide to ELISA Development
The ideal ELISA developed in-house should be
G Sensitive: the method is capable of measuring the ana-
lyte at a sufﬁciently low concentration for the intended
application of the test.
G Speciﬁc: the method has negligible cross-reactivity to
molecules structurally similar to the analyte that may
be present in the samples.
G Simple: the method is easy to perform and gives quick
G Stable: the reagents used in the assay are thermostable,
and the analytical performance is robust.
G Safe: the reagent components used in the method are
not harmful and require no speciﬁc handling.
Key Steps of ELISA
ESTABLISH THE REQUIREMENTS
The ﬁrst stage of any design process is the deﬁnition of the
requirements, and for ELISA, this includes the technical
constraints, raw materials, and analytical performance
expectations. The requirements must answer the following
G What is to be measured (analyte)?
G What are the sample matrices (serum, plasma, cell
lysates, ascites, etc.)?
G What is the detection limit required?
G How speciﬁc must the ELISA be?
G What is the measurement range?
G How accurate must the ELISA be?
G What is the detection mode (colorimetric, ﬂuorescence,
or chemiluminescence) and which plate reader(s) can
G What are the requirements for stability of the reagents,
standards, calibrators, controls, and samples?
Based on the answers to the questions above, a prototype
ELISA method is ﬁrst developed to establish the proof of
concept, i.e., the feasibility of the project. Optimization of
the component formulations and protocol steps comes later.
UNDERSTAND THE ANALYTE
The analyte is the target that the ELISA needs to detect
and in most cases quantify. Common analytes are proteins
(including antibodies), smaller naturally occurring sub-
stances such as steroid hormones, and drugs and other syn-
thetic compounds. It is particularly important to
understand the characteristics of the analyte in the context
of ELISA method development. The characteristics of
analytes can be found through literature and patent
reviews. Such information includes structure, molecular
weights, isoelectric point (pI) value, antigenicity, solubil-
ity, and thermal stability.
Research the behavior of the analyte in the type of sam-
ple under test. For example in blood, check if the analyte is
bound to a carrier protein, such as albumin.
Two important considerations for ELISA are the
immunogenicity of the analyte: its ability to provoke an
antibody response; and antigenicity: its ability to bind to
antibodies. These are two different but related characteris-
tics. This indicates how difﬁcult it will be to acquire or
generate suitable antibodies. Understanding the size and
the spatial relationship of the epitopes (antigenic determi-
nants) on the analyte is very useful in the selection of anti-
bodies and can greatly affect assay performance. Linear
epitopes consist of a continuous sequence of amino acids
in protein analytes, and conformational epitopes exist
when discontinuous sections of amino acids form an anti-
genic determinant. The distribution and variability of epi-
topes are also informative for ELISA reagent formulation.
When ELISAs are required to detect speciﬁc antibodies, it
is necessary to deﬁne the class or subtypes of the antibody
to be measured. Sometimes, ELISA is used to measure
Small molecules (called haptens) can elicit an immune
response only when attached to a large carrier, usually a
protein, although the carrier may be one that by itself does
not elicit an immune response. Once antibodies have been
generated, the small-molecule hapten may also be able to
bind to the antibody.
For research applications, the analyte to be measured
can be a recombinant form of the natural protein. What is
the difference in antigenicity, stability, binding afﬁnity,
and reactivity between the recombinant protein and its
The expected analyte concentration range likely to be
encountered in the sample matrix of choice determines the
detection limit and the measurable range that should be
achieved in a validated method.
CHOOSE A SUITABLE ASSAY FORMAT
Four formats are frequently used for ELISA: immunomet-
ric (sandwich), competitive, indirect, and immunocapture.
The selection of ELISA format depends on the availability
of key reagents, and the assay sensitivity and dynamic
range required for the application.
The immunometric (sandwich) format usually uses two
antibodies that preferably bind to different sites on the
antigen. The primary antibody, which is highly speciﬁc
for the antigen, is attached to the microtiter plates. The
samples containing the analyte are then added, followed
by addition of the detection antibody, which is conju-
gated to an enzyme. As a result the analyte is “sand-
wiched” between the two antibodies. Sometimes, to
increase sensitivity, multiple antibodies can be attached
to the microtiter plates for capture. In the ﬁnal step of
the assay, the signal generated is proportional to the
amount of target analyte present in the sample. Com-
pared to other formats, the immunometric format is
more sensitive, precise, and robust. Not surprisingly
therefore, it is the most commonly used. However, the
analyte molecule needs to be large enough (with a molec-
ular weight greater than 6000Da) to have two separate
antigenic sites (see Fig. 1).
384 The Immunoassay Handbook
The competitive format is used for analytes with a low
molecular weight. It relies on a single antibody speciﬁc for
the analyte. For optimal results, afﬁnity puriﬁed antibodies
are preferred. Compared to the immunometric format, the
sensitivity of competitive assay is more deﬁned by the
equilibrium constant of the antibodies, precision of signal
measurement, and level of nonspeciﬁc binding. Develop-
ment and validation of a competitive ELISA require con-
siderable expertise in reagent characterization and method
development. In general, a competitive ELISA is not as
sensitive as a sandwich ELISA, has a narrower working
concentration range, and is more susceptible to matrix
effects. The timing of the various incubation steps is more
critical in the design of a competitive ELISA. See Fig. 2.
The indirect format is used for detection of speciﬁc anti-
bodies, e.g., antiviral IgG. The format uses antigen coated
onto the plate to capture antibodies, and then, the captured
antibodies are detected by species-speciﬁc anti-IgG
or IgM. The indirect format is also susceptible to nonspe-
ciﬁc binding. The purity and speciﬁcity of the antigen to
be coated on the microtiter plates are very critical to the
speciﬁcity of the ELISA method. See Fig. 3.
The immunocapture format is also designed for detection
of speciﬁc antibodies, usually IgM antibodies. This format
uses animal anti-IgM to capture the IgM in the sample,
then a speciﬁc enzyme-labeled antigen or antigen paired
with enzyme-labeled speciﬁc antibody is used to detect the
IgM of interest. This format requires a speciﬁc antigen
with high purity for labeling or bridging to the enzyme-
labeled antibody. See Fig. 4.
For research purposes, the ELISA method is usually het-
erogeneous and requires a solid phase that facilitates sepa-
rating the bound from the unbound analyte and matrices.
Immobilizing or coating is the process used to attach the
speciﬁc antibodies or antigens onto the solid phase that
FIGURE 2 Competitive format.
385CHAPTER 5.1 Practical Guide to ELISA Development
captures analytes in the later steps. Microtiter plates, which
are easy to handle and process, are the usual solid phase for
ELISA. They are available either in the form of 96-well
plates or 8- or 12-well strips. Due to its high capacity for
protein binding, polystyrene is the usual plastic chosen.
Flat-bottomed wells are recommended for spectropho-
tometer readings, round-bottomed (U-shaped) wells are
more suitable for visual assessment.
ANTIBODY OR ANTIGEN REAGENTS
Afﬁnity is the foundation of all immunoassay develop-
ment, including ELISA. In general, the binding process
between antigen and antibody can be attributed to ener-
getic factors, such as van der Waals forces, hydrogen
bonds, and ion pairs. But the intrinsic afﬁnity for anti-
body–antigen binding is affected by many factors, includ-
ing temperature, time, pH, ionic strength, detergent
type and concentration, and the concentration of other
macromolecules. Typically, the goal in the feasibility
phase of ELISA development is to achieve high assay
sensitivity by selecting workable antibody pairs and by
maximizing the antibody-antigen binding afﬁnity. Ulti-
mately, in this phase, we want to identify the best anti-
body or antibody pair and create an environment in
which the antibody and antigen are most adapted to each
FIGURE 3 Indirect format.
386 The Immunoassay Handbook
Antibodies and antigens are key biologicals in ELISA
design. For in-house ELISA development, the antibodies
can be from donation, purchased, or generated by the
researchers. Many kinds of antibodies can be used in ELISA,
but monoclonal and polyclonal IgG antibodies are the most
common. Afﬁnity and speciﬁcity are the key characteristics
for antibody selection. Antibody afﬁnity is determined by
comparing the rate of the antibody–antigen formation to
the rate of dissociation. Often, the afﬁnity constant deter-
mines the potential sensitivity of the assay. It is recom-
mended that the afﬁnity constant be greater than
1010–1011 L/mol and ideally 1012 L/mol. Antibodies with
higher afﬁnity may be less susceptible to interferences (from
the environment, instrument, or different detection tech-
niques) and typically these antibodies provide more robust
results. Binding afﬁnity is inﬂuenced by the antigen: those
with multiple repeating epitopes naturally generate higher
avidity in binding than antigens with a single epitope. An
afﬁnity constant <108 L/mol is usually not suitable for an
ELISA. Many companies do not sell their best antibody
clones but retain them for their kits. This is the reason that
in-house assays sometimes cannot match the performance
of the commercial kits for the same analyte.
To ensure detection speciﬁcity, analyte-speciﬁc anti-
bodies should be used. Consult manufacturers’ data sheets
for information on cross-reactivity. However, highly spe-
ciﬁc antibodies may not detect all the antigen isoforms,
and weak afﬁnity for certain isoforms may result in assay
drift and imprecision. To achieve the right balance, it is
important to consider how the assay will be used.
FIGURE 4 Immunocapture format, using a test for anti-hepatitis A virus IgM as an example.
387CHAPTER 5.1 Practical Guide to ELISA Development
In assays in which antigen is employed as a reagent, i.e.,
competitive, indirect, and immunocapture assays, it is
important to understand the biochemistry of the antigen,
and how it behaves with respect to the antibodies it will
bind with in the assay.
The antibody lies at the heart of an ELISA. Identifying the
best antibodies and the correct roles for these antibodies
(capture vs detection) is a critical balancing act. The cap-
ture antibody typically has high afﬁnity and speciﬁcity for
the analyte in the sample, whereas detection antibody may
contribute less to analyte speciﬁcity. This is particularly
true in ELISA methods with multiple wash steps prior to
the addition of the detection antibody. As discussed above,
the antibody-antigen binding afﬁnity is usually what
determines ELISA sensitivity and, as a result, researchers
need to prudently select antibody pairs in order to avoid
any potential cross-reaction by structurally related
The choice of antibody pair will depend on the antigen
to be detected and the availability of antibodies to differ-
ent epitopes on the antigen. A large number of monoclo-
nal antibodies (mAb) and polyclonal antibodies (pAb) are
available commercially and can be identiﬁed quickly by
searching on websites. Alternatively, new speciﬁc antibod-
ies may be created to recognize the antigen of interest.
Both pAb and mAb work well for ELISA. Crude antibody
preparations such as serum or ascites ﬂuid are sometimes
used for ELISA, but the impurities present may increase
background. To obtain antibodies with the greatest speci-
ﬁcity, they can be afﬁnity puriﬁed using the immobilized
Either mAb or pAb may be used as the capture and
detection antibodies in sandwich ELISA systems. PAbs are
often used as the capture antibody to pull down as much of
the antigen as possible. Then, a mAb is used as the detect-
ing antibody to provide speciﬁcity. It is important to note
the differences between a mAb and a pAb.
MAbs are derived from a single cell line and have an
inherent monospeciﬁcity toward a single epitope that
allows ﬁne detection and quantitation of small differences
in antigen. Because of this, they provide high speciﬁcity—
but it is at the expense of sensitivity, since only one anti-
body molecule can bind to the antigen. They are valued
for their speciﬁcity, purity, and consistency, which result
in lower background.
On the other hand, pAbs are derived from different
B-cell lines and generate a variety of responses to multi-
ple epitopes with different afﬁnities within a deﬁned
antigen. This creates a higher sensitivity because multi-
ple antibodies are binding to a single antigen molecule.
Polyclonal antibodies are less expensive and less time
consuming to produce. Each type of antibody has pros
and cons. One downside of pAbs is that they have a
higher risk of nonspeciﬁc binding. But on the other hand,
they show better functional afﬁnity and cooperativity in
multivalent binding across different epitopes. Therefore,
they may demonstrate excellent binding overall, since
they have adhered to a number of different sites on a
complex immunogen or antigen. In fact, for assays
requiring a broad spectrum of speciﬁcity to large molec-
ular weight antigens, pAbs are the clear choice. Addition-
ally, pAbs are more suitable to be used as detection
antibodies. One problem is that there is a ﬁnite availabil-
ity of pAbs derived from a particular source, and this
makes it difﬁcult to achieve continuity of assay perfor-
mance after the original source runs out. It is important
to select the highest afﬁnity and most speciﬁc antibody
available. But again, balance is the key: if taken too far,
this strategy can increase the susceptibility of the assay to
heterogeneity of analytes.
When it comes to mAbs, the single-epitope speciﬁcity
makes these antibodies more vulnerable to structural or
conformational changes in the epitope. To overcome this,
a cocktail of mAbs is sometimes used in an assay. In gen-
eral, mAbs make excellent primary antibodies in immuno-
metric immunoassays; and in competitive assays for drugs,
hormones or other small analytes, mAbs are the best choice
for quantitative measurement. Additionally, for assays that
require good reproducibility, mAbs may be a better choice
When determining the right balance in antibody pairs,
it is important to remember that antibody functions in
ELISA tests are switchable. For example, some antibod-
ies are better suited in a capture role but can also per-
form well in a detection role with speciﬁc coupling
chemistry. Finding the correct antibody pair and choos-
ing each antibody’s role is a critical early step in ELISA
An important consideration in designing an immuno-
metric ELISA is that the capture and detection antibodies
must recognize two nonoverlapping epitopes in the sand-
wich format. When the antigen binds to the capture anti-
body, the epitope recognized by the detection antibody
must not be obscured or altered. Capture and detection
antibodies that do not interfere with one another and can
bind simultaneously are considered a matched pair and are
suitable for developing a sandwich ELISA. Preparing a
“self-sandwich” ELISA assay, in which the same antibody
is used for the capture and detection, can limit the dynamic
range and sensitivity of the assay.
Besides determining which antibody should be capture
antibody and which should be detection antibody, the
optimum antibody concentrations for both the capture
and detection antibodies are also critical. A practical
approach is to coat the ELISA plate with several dilutions
of both antibodies that will be used as part of the sandwich
assay. Add the analyte to be measured at high, low, and
zero concentrations. Use both of the antibodies at several
concentrations as a secondary antibody. Determine the
Absorbance Units (AUs) that yield the maximum signal-
to-noise ratio or the greatest difference between the high
and low analyte concentrations with the lowest variability.
As a ﬁrst step, these are the conditions that could be
selected for the antibodies.
Antibodies play fundamental roles in determining the
sensitivity and dynamic range of ELISA. This is due to the
actual antibody afﬁnity for the analyte after selection and
optimization of the reagents for the antibody and antigen
reaction taking place. If after attempting to develop the
assay the sensitivity is still not in the desired range, differ-
ent antibody pairs will need to be evaluated.
388 The Immunoassay Handbook
Microtiter Plate Coating
The attachment of the antibodies or antigens to the sur-
face of the microtiter wells involves non-covalent bonds
between the hydrophobic regions of the protein and the
nonpolar plastic surface. Proteins including antibodies are
often readily immobilized, but the coating efﬁciency varies
from protein to protein. Simple non-covalent adsorption
of antibodies to microtiter wells is often recommended for
in-house developed ELISA since it is an easy process to
use. The coating buffer must be free of any protein other
than the coating protein.
Understanding the interfacial interactions between the
solid surface and the coated protein is profoundly impor-
tant during the design of the solid phase. There are many
factors to consider when coating the protein to the solid
G Surface materials.
G Coating chemistry.
G Antigen or antibody characteristics.
G Incubation time.
G Incubation temperature.
Immobilization can alter the way antibodies and antigens
function and can potentially cause the antigen to lose criti-
cal epitopes. This effect is greater when protein is coated at
a low concentration. However, coating at a high concen-
tration may result in more aggregation. It is important to
note that antibodies and antigens have different physio-
chemistries (e.g., hydrophobicity, isoelectric point, glyco-
sylation, and molecular weight). As a result, the coating
conditions should be adjusted to each antibody or antigen.
In general, protein-binding capacity is proportional to
the hydrophobicity on the solid surface. A highly hydro-
phobic surface may cause more structural disruption to a
coated macromolecular protein compared to the same
protein that is not coated. Hydrophobic coating increases
the capacity, and stronger blockers may be required to
mask nonspeciﬁc binding. Usually, most of a protein’s
hydrophilic residues are located at the outside, and most of
the hydrophobic residues orientated toward the inside.
Partial denaturation of some proteins results in exposure
of hydrophobic regions and ensures ﬁrmer interaction
with the plastic.
While conformation is highly protein speciﬁc, covalent
coating may cover or alter the antigen-binding site of the
antibody. Several problems arise from passive adsorption,
including desorption, improper orientation, denaturation,
poor immobilization efﬁciency, and nonspeciﬁc binding
along with the target molecule. Alternatively, antibodies can
be attached to a microplate through the Fc region using Pro-
tein A-, G-, or A/G-coated plates, which orients them prop-
erly and preserves their antigen-binding capability. Precoated
plates bind selectively to the desired target proteins, minimiz-
ing any contamination from other molecules that are present
in the preparation. It is important to ensure that the coating
solution is free of detergents because detergents often com-
pete for binding and cause low and/or uneven binding. In
addition, the coating buffer should contain no other proteins
that might compete with the target antigens for attachment
to the plastic solid phase. High purity of the antigen or anti-
body to be coated will increase coating efﬁciency as well as
assay speciﬁcity. As a rule of thumb, about 0.5–1µg of IgG
may be bound per cm2 in an efﬁcient coating process. For
competitive assays, a lower coating concentration usually is
chosen to ensure that the antibody is the limiting factor. The
following factors need to be considered during coating:
G Diffusion coefﬁcient of the attaching molecules.
G Ratio of the surface area being coated to the volume of
the coating solution.
G Concentration of the substance being adsorbed.
The range of protein concentrations for coating is usually
within 1–10µg/mL and with a volume of 50–100µL. How-
ever, the concentration should be titrated to achieve
optimum antibody binding allowing for orientation and
steric effects. The coating temperature is often either
37°C for 1–3h or 4°C overnight. Increasing the tempera-
ture may shorten the incubation time, but some antigens
are unstable at higher temperatures.
The following coating buffers are often used:
G 50mM carbonate, pH 9.6,
G 20mM Tris–HCl, pH 8.5,
G 10mM phosphate-buffered saline, pH 7.2.
It is advisable to use a buffer with a pH value 1–2 units
higher than the pI value of the protein being attached,
avoiding protein precipitation during the coating. The
amount of 50–500ng per well has been found valid for a
variety of proteins in 50µL volumes.
Blocking is another essential step in ELISA, particularly
when reducing the nonspeciﬁc binding of primary or sec-
ondary antibodies to the solid surface of microtiter plates
and nonspeciﬁc binding with low afﬁnity from sample to
solid surface. Blocking the unoccupied sites on the surface
of the well can reduce the amount of nonspeciﬁc binding of
proteins during subsequent steps in the assay. Therefore, it
can also increase assay sensitivity and speciﬁcity. Here
again, no single blocking agent is ideal for every occasion
because each antibody–antigen pair has unique character-
istics. A variety of blocking buffers ranging from nonfat
milk to highly puriﬁed proteins have been used to block
unreacted sites. Empirical testing is necessary. The proper
choice of blocker for a given assay depends on the antigen
itself and on the type of enzyme conjugate to be used. For
example, with applications using an alkaline phosphatase
(ALP) conjugate, a blocking buffer in TBS should be
selected because PBS interferes with ALP. The ideal block-
ing buffer will bind to all potential sites of nonspeciﬁc
interaction, eliminating background altogether, without
altering or obscuring the epitope for antibody binding.
Separation and Washing
The sample may contain other biological or chemical con-
stituents that can interfere with the subsequent signal
389CHAPTER 5.1 Practical Guide to ELISA Development
generation stage that need to be washed away after capture
of the analyte. After incubation with the enzyme-labeled
antibody, the unbound enzyme must also be removed by
washing to reduce the assay nonspeciﬁc binding. Washing
is one of the critical steps of ELISA. It can be easily and
reproducibly carried out with inexpensive washing devices,
which are manual, semiautomated or fully automated.
The washing buffer usually contains 0.15mol/L NaCl
and 0.05% Tween 20. The frequency of washing depends
on the step. Three to ﬁve washing steps with 0.5mL of
washing buffer are recommended after each incubation
stage. Slamming the inverted plate onto a wad of tissues
is a good way of removing any residual droplets of wash
solution, and this can improve the precision of some
assays. But it is important to keep the wells moist at all
times between each washing step. If they dry out, the
washing may not be effective, leading to high
For an in-house ELISA, it is simplest to use an off-the-shelf
commercial enzyme-labeled antibody for the signal genera-
tion. This is known as a secondary antibody because it is
not speciﬁc for the antigen under test but for the detection
antibody. The choice of secondary antibody depends upon
the species of animal in which the primary antibody was
raised (the host species). For example, if the primary anti-
body is a rabbit antibody, the secondary antibody could be an
anti-rabbit from goat, chicken, etc. but not rabbit. If a sec-
ondary antibody causes high background in an assay, a sec-
ondary antibody from another species may improve results.
An alternative option to reduce background is to use a
secondary antibody that has been pre-adsorbed to serum
proteins from other species. This pre-adsorption process
removes antibodies that have the potential to cross-react
with serum proteins, including antibodies. Antibodies
for ELISA are typically used as diluted 1/100–1/500,000
starting from a 1mg/mL stock solution. The optimal dilu-
tion of a given antibody with a particular detection system
must be determined experimentally. Antibody dilutions
are typically made in the wash buffer containing a blocking
agent. The presence of a small amount of blocking agent
and detergent in the antibody diluent often helps to mini-
Many labeled secondary antibodies are commercially
available. Using secondary antibody, the sensitivity is
increased because each primary antibody contains several
epitopes that can be bound by the labeled secondary anti-
body, allowing for signal ampliﬁcation. However, it can be
susceptible to nonspeciﬁc binding due to the presence of
the secondary antibody and the extra incubation step.
CHOICE OF ENZYME
The most commonly used enzymes are horseradish per-
oxidase (HRP) and ALP. These enzymes can be detected
further with the use of colorimetric, chemiluminometric,
and ﬂuorogenic techniques. However, some of these tech-
nologies require special instrumentation. An absorbance
microtiter plate reader is the most practical. The results
are shown through color development.
A large selection of substrates is available for performing
the ELISA with an enzyme conjugate. The choice of sub-
strate depends upon the required sensitivity level and the
instrumentation available in the individual laboratory for
Labeling is the process to conjugate the antigen or anti-
bodies to an enzyme that can generate signal for detection
in the presence of substrate and the corresponding system
for measuring the captured analyte. The enzyme may be
conjugated directly to the primary antibody or introduced
through a secondary antibody that recognizes the primary
antibody. However, for research purposes, direct labeling
is not recommended because it is labor intensive and time
consuming; it also needs special expertise.
HRP is the most commonly used enzyme conjugated to
antibodies in ELISA. It is a 44kDa glycoprotein with four
lysine residues for conjugation to the target molecule. It
can produce a colored, ﬂuorimetric, or luminescent deriv-
ative when incubated with the appropriate substrate. It
also has a high turnover rate that allows generation of
strong signals in a relatively short time span. HRP is active
over a broad pH range with respect to its substrate: from
pH 4.0 to 8.0. HRP is more stable in 0.1M citrate than
0.1M phosphate buffer. High-molar phosphate buffer can
be particularly damaging to HRP at low pH. Nonionic
detergents can affect the stability of the enzyme. There are
a variety of substrates available for HRP, such as tetra-
methylbenzidine (TMB), 3,3′-diaminobenzidine (DAB),
and 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)
ALP is a 140 kDa protein that catalyzes the hydrolysis of
phosphate groups from a substrate molecule, resulting in a
colored or ﬂuorescent product or the release of light as a
byproduct. It has optimal enzymatic activity at a basic pH
(pH 8–10) and can be inhibited by EDTA. Mg2+ is essen-
tial for enzymatic activation. As a label for ELISA, ALP
offers distinct advantages over other enzymes. The enzyme
activity is not affected by exposure to antibacterial agents,
such as sodium azide, which is in the formulation of many
ELISA buffers. Because its reaction rate remains linear,
detection sensitivity can be improved by simply allowing a
reaction to proceed for longer. Nonionic detergents
appear to have no effect on enzyme activation. The most
common ELISA substrate for alkaline phosphatase is the
chromogen p-nitrophenyl phosphate (PNPP), which is
available in several formats. For example, Fischer Scien-
tiﬁc provides PNPP as dry crystalline powder, single-use
tablets, a stable substrate solution and as a kit containing
substrate solution and diethanolamine buffer (www.ﬁsch-
Chemiluminescent substrates are suitable for both
enzymes. Chemiluminescent substrates differ from other
substrates in that the signal is a transient product of the
reaction that is only present while the enzyme–substrate
reaction is occurring. This is in contrast to substrates
that produce a stable, colored product, which persist in
the well after the enzyme–substrate reaction has termi-
nated. In a chemiluminescent ELISA, the substrate is the
390 The Immunoassay Handbook
limiting reagent in the reaction; as it is exhausted, light
production decreases and eventually ceases. Therefore, a
well-optimized procedure using the proper antibody
dilutions will produce a reproducible output of light. If
the antibody is not diluted sufﬁciently, too much enzyme
is present, and the substrate is used up quickly, so a stable
output of light will never be achieved. This is the single
greatest cause of variability in chemiluminescent ELI-
SAs. To avoid this problem, it is crucial to optimize the
amount of antibody used for detection. Antibody suppli-
ers typically suggest a dilution range for using their anti-
body in an ELISA. This dilution range is often appropriate
for ELISAs detected with a relatively insensitive chro-
mogenic substrate, but a much greater dilution is gener-
ally required for optimum performance with a sensitive
In competitive immunoassays, labeling an antigen may
alter its epitopes and affect the antigenicity for the detec-
tion antibody, but the converse can also be true and label-
ing may augment the recognition. Typically this is due to
bridge recognition, in which the labeling is directed to the
same position that the hapten was originally conjugated to
on the immunogen. The following factors need to be con-
sidered when conjugating antibodies or antigens:
G Size of enzyme.
G Ease of conjugation.
In general, HRP is ideal in many respects for these appli-
cations because it is smaller, more stable, and less expen-
sive than ALP. For both enzymes, the substrates can be
made up in buffer stored frozen in well-sealed vials and
then thawed for use. The substrate needs to be stable.
Finally, strong acids or bases stop enzymatic activity by
quickly denaturing enzymes. Sodium azide is a potent
inhibitor of HRP, whereas EDTA inhibits ALP by the
chelation of metal cofactors.
The generation of color is controlled by the addition of
a stopping reagent, which must be added at an accurately
controlled volume, since photometric readings are affected
if the total volume of reactants varies. Color is measured
using a spectrophotometric plate reader. The product of
substrate catalysis by the enzyme is measured by transmit-
ting light of a speciﬁc wavelength through the solution and
measuring the amount of absorption of that light. The
correct ﬁlter wavelengths should be selected for spectro-
The design of ELISA is a science, because many experi-
ments need to be carried out to achieve the best results
with a unique set of biological, chemical, and physical ele-
ments. It is also a craft, in which practitioners evolve their
own sequence of activities, based on their experiences and
those who teach them. But it is also an art, because the
most experienced ELISA developers do not follow a ﬁxed
protocol, but view the ﬁnal ELISA as a unique integrated
system, transcending the many elements involved to
achieve a blend of goals that is deﬁned by the assay creator.
The fundamental objectives of any ELISA development
(1) Identify the correct antibody or antigen.
(2) Create an optimum environment for assay kinetics
and binding afﬁnities.
(3) Provide consistent results with maximum assay sen-
sitivity and speciﬁcity.
To meet these objectives, researchers need to balance
many factors—all within the constraints imposed by qual-
ity, cost, and time.
KINETICS AND THE INCUBATION TIME
Once the formulations of the reagents have been estab-
lished, assay performance is optimized. By carrying out
simple kinetic experiments, the relationships between
incubation times and signal generation intensity are estab-
lished for a range of reagent concentrations. The goal is to
achieve a high signal-to-noise ratio, not simply to maxi-
mize the signal. Sensitivity and precision are key goals.
Practical considerations also apply, for example, the incu-
bation time needs to be acceptable. Faster assays may be
achieved with higher concentrations of conjugate (enzyme-
labeled antigen or antibody), but the cost and availability
of the conjugate are another consideration.
There are usually trade-offs between desirable perfor-
mance characteristics, for example, a short incubation time
may result in weak signal generation, and a long incuba-
tion time may favor nonspeciﬁc signal generation. The
incubation time optimization often starts with observation
of the impact of time and temperature on signal intensity
and nonspeciﬁc binding. Adjusting incubation time can
often be beneﬁcial to reducing nonspeciﬁc binding. If this
does not achieve the desired results, using more speciﬁc
mAbs should result in a higher signal-to-noise ratio.
Immobilization of one reactant to the solid phase may
mean it takes longer to reach equilibrium than if the reac-
tant was in solution. Assays with requirements for a fast
turnaround time, such as cardiac markers, typically use high
concentrations of conjugate to speed up the assay reaction.
Cross-reactivity often decreases with incubation time
and is minimal when equilibrium is reached. Increas-
ing the incubation temperature may also decrease
In kinetics, overall balance is important. Kinetics should
be optimized in combination with other factors such as
reaction volume, number of steps, conjugate mass, and
matrix effects. For instance, a smaller volume may be
favorable for a reaction, but a relatively larger volume
would create less variation. Kinetics are also dependent on
pH, ionic strength, and temperature. Ion content and pH
in particular can affect assay kinetics and may affect some
mAbs more than others.
Another consideration: sandwich and competitive assays
have different thermodynamic equilibriums. Sandwich
formats generally require shorter incubation times because
it is acceptable to use higher capture antibody and conju-
gate concentrations. Meanwhile, competitive formats
require careful consideration of reagent inputs. In addi-
tion, as antibody inputs are adjusted, it is important to con-
sider the high-dose hook effect in sandwich assays.
391CHAPTER 5.1 Practical Guide to ELISA Development
Buffers are the backbone of ELISA. Multiple factors
should be considered in buffer formulation such as the
buffer system (Tris, phosphates, HEPES, or MES, etc.),
ionic strength, pH, salt, detergent, proteins, blocking
agents, preservatives, and other additives. Reagent formu-
lation is unique to each ELISA method; and while using
reagents off the shelf may shorten development time; this
practice may also lead to potential problems because the
reagents are not optimized.
It is particularly important to consider antigen charac-
teristics. In general, the use of detergents may help reduce
nonspeciﬁc binding. But the proper use of a detergent
depends upon the speciﬁc antigen. An antigen with epitope
made up of continuous segments of polypeptide chain (lin-
ear epitope) can better stand detergent and vigorous for-
mulation compared to a conformational epitope, which is
made up of a juxtaposition of multiple segments from dif-
ferent parts of the protein sequences. In fact, the presence
of a conformational epitope may limit or inhibit the use of
a detergent, or high salt, and require a speciﬁc pH range.
In addition, matching the surface charge plays an impor-
tant role in speciﬁcity, especially for highly charged pro-
tein antigens. Charge is a strong force in non-covalent
antibody–antigen binding. Therefore, neutralizing the
charge can help to stabilize this kind of antibody–antigen
complex and lead to better recognition. Another consider-
ation is antigen size. The binding site can be structurally
classiﬁed into three major types: cavities, grooves, and pla-
nar sites. These correspond to the size and shape of the
antigen being bound. Small molecules or short peptides
typically bind in a pocket or groove lying between the
heavy and light chain variable regions, and there may only
be contact between one to two amino acids in the antibody
molecule and the epitope. As a result, small antigens may
be very sensitive to subtle changes in buffer formulation or
optimization. Large protein antigens bind in the planar
site and may contact 15–20 amino acids in the binding site.
Here, buffer optimization may result in better surface
complementarity, which is also very important to the
mobility, accessibility, and antigenicity of an antigen. Flex-
ibility of the antigen will allow its epitope to more readily
assume the best and most avid conﬁguration in the anti-
Signal-to-noise ratio is the best indicator for the correct
selection of blocker(s). And as with the other steps, there
is a question of balance. Over-blocking may alter or
obscure the epitope for antibody binding. In addition,
excessive blocker could mask antibody–antigen interac-
tions, or inhibit the enzyme, and result in a reduction of
the signal-to-noise ratio.
REAGENT FORMULATION AND
The goals in developing an ELISA assay are
G To achieve the best signal-to-noise ratio for the sensi-
tivity level desired.
G To be able to measure the antigen or antibody over a
G To have a robust, reproducible assay for the sample
Therefore, optimal concentrations of each assay reagent
must be established empirically. The signal-to-noise
ratio is the ratio of the signal level of a sample containing
the target analyte to the level of noise. Noise is the stan-
dard deviation (SD) of the signal when a sample from
which the analyte is absent is repeatedly tested. As the sig-
nal-to-noise ratio increases, the assay becomes more effec-
tive at measuring small amounts of antigen.
There are two approaches to increasing the signal-to-
noise ratio: reduce the noise or increase the signal for a
given analyte concentration. By adjusting the reagent dilu-
tions, the optimum signal-to-noise ratio may be achieved
One way to establish the optimal dilutions is using
checkerboard titration, also called a two-dimensional
serial dilution. A checkerboard titration is a single exper-
iment in which the concentration of two components is
varied in a way that will result in a pattern. This design
permits analysis of different concentrations of the two
reagents in each well to obtain the highest signal-to-noise
Yet another factor that needs to be taken into account is
sample volume. Sample has an evident impact on reagent
formulation for assay development. Ideally, sample volume
should be deﬁned by minimal interference and matrix
effect. Assays vary in their susceptibility to this type of
effect. The antibody–antigen reaction occurs at the solid–
liquid interface, so the exact reaction volume is difﬁcult to
determine. Large sample volumes may increase assay sensi-
tivity, but they may also result in higher matrix effects and
lower linearity. Sample matrix effects can be minimized by
using a low ratio of the sample compared to the assay
reagent in the incubation. However, this reduces the signal
level and potentially the signal-to-noise ratio, reducing the
sensitivity of the assay. Alternatively, increasing protein and
ionic concentration, as well as buffering capacity, may also
mitigate matrix effects from samples.
Hardware and Software
The instrumentation to measure the signal reﬂecting the
extent of antigen–antibody binding is determined by the
conjugated enzyme and substrate combination. In ELISA
methods, a soluble substrate is used to generate a signal in
solution. Enzyme-labeled reagents may be detected using
chromogenic, chemiﬂuorescent, or chemiluminescent
substrates, using spectrophotometer, ﬂuorometer, or
luminometer, respectively. Where the detection is colori-
metric, a spectrophotometric plate reader is used.
For absorbance measurement, especially with manual
handling, dual-wavelength is commonly used. In dual-wave-
length detection, the ﬁrst measurement usually corresponds
392 The Immunoassay Handbook
to the maximum absorbance, and the second measurement is
taken at a wavelength near the baseline as a blank reference
value for each well, which includes not only baseline absor-
bance but also absorbance due to exogenous materials that
could randomly adhere to the outside of the wells and con-
tribute to imprecision. The microplate readers conveniently
subtract the second absorbance reading from the ﬁrst mea-
surement and relate the difference to the analyte concentra-
tion (Diamandis et al., 1996).
The instrument used to read the output of the ELISA
should be tested initially for both linearity and perfor-
mance. Instrument performance should be regularly tested
and recalibrated according to the manufacturer’s
CALIBRATION CURVE FITTING
The computerized ﬁtting of a calibration curve is carried
out using a commercial product. A printout of the curve
indicates whether there are any biases between the com-
puted curve and the calibrator signal levels. This subject is
well covered in the chapters CALIBRATION CURVE FITTING
and IMMUNOASSAY TROUBLESHOOTING GUIDE.
STANDARDIZATION AND CALIBRATION
Known concentrations of analyte are used to provide cali-
bration curves against which unknown sample concentra-
tions can be ascertained. See the chapters STANDARDIZATION
AND CALIBRATION and IMMUNOASSAY TROUBLESHOOTING
QUALITY CONTROL AND VALIDATION
The purpose of quality control is to independently verify
the reproducibility and, if there is a standard, the accuracy,
of the results obtained using the ELISA method. Control
samples can be from patients or made by spiking and pool-
ing. Ideally, the analyte concentration in the control sam-
ples should have been determined by another validated
method. Spiked controls are created by adding a known
concentration of the standard analyte into the usual sample
matrix and used in each experiment to track method per-
formance. Spiked controls can be used to determine assay
performance based on calculating the percent dose recov-
ery. The following features need to be evaluated in choos-
ing an antigen for quality controls:
ELISA intended for research use needs to meet the
required speciﬁcations for method performance. Before
designing the ELISA method, it is advisable to deﬁne the
achievable speciﬁcations. By setting the bar for the new
method too high, there is a risk that development will be
thwarted rather than encouraged. Major parameters to
evaluate during the validation are
G Sensitivity and speciﬁcity.
G Imprecision and repeatability.
G Linearity and stability.
G Cross-reactivity and interference.
ELISA Tips and
FACTORS AFFECTING ELISA RESULTS
G Temperature: temperature variation will affect anti-
body-binding afﬁnities. Variation is most likely
between the corners and center of a microtiter plate. A
stack of plates is more vulnerable, between the corners
of the top and bottom plates and the center of the
stack. Test for temperature effects by assaying repli-
cates of one control or sample pool across an entire
plate. Calculate the mean and SD of the optical densi-
ties (ODs) from all the wells and then place each well
signal level in one of the following groups: ≥2 SD, −2
to −1 SD, −1 to 0 SD, 0 to +1 SD, 1–2 SD, and >2 SD.
Plot the distribution of signal levels across the plate by
marking each well position with −−− (≥2 SD), −− (−2 to
−1 SD), − (−1 to 0 SD), + (0 to +1 SD), ++ (1–2 SD),
and +++ (>2 SD). Effects due to temperature gradients
in the incubator will show up clearly. If patterns show
up either increase the incubation time or improve the
plate incubator. Temperature effects have characteris-
tic patterns, usually with the greatest difference from
the center to the corners. This same test may also show
up other characteristic patterns, such as manifold
effects affecting the efﬁciency of plate washers (from
the center line to the sides) or drift (from the ﬁrst to
the last well).
G Time: accuracy of time is a critical matter for a con-
sistent testing result. Therefore, it is recommended
to always use the same procedure and follow the
same order for addition of reagents, taking the same
(short) time for pipetting across the plate. Time
effects show up as drift from the ﬁrst to the last well
if a sample pool is assayed in every well. Drift can be
reduced by increasing the incubation time or improv-
ing the consistency of pipetting times for consecutive
G Movement: shaking during incubation is preferable.
Plate rotation is better than leaving plates stationary. It
eliminates viscosity effects and reduces the likelihood
of temperature gradients.
G Receive good training.
G Follow the manufacturer’s directions.
G Be careful in sample preparation and avoid samples
with lipemia, hemolysis, and particulates.
G Pipette with accuracy.
1. Avoid leaving the pipette on the side of the plate.
2. Avoid frothing on addition of samples.
3. Keep the same order and pace for pipetting.
393CHAPTER 5.1 Practical Guide to ELISA Development
4. Finish pipetting one plate within 2 min, one plate
at a time.
G Be careful in handling conjugates and make fresh work-
G Be careful in handling positive samples; do not cause
cross-contamination of negative wells or samples.
G Carry out washing thoroughly, especially where ana-
lytes are in cell lysates, or require extraction using
reagents containing SDS or other denaturing reagents
that may interfere with the assay. Wash with PBS
G Make sure the plate reading is at the correct
G Use good quality water.
G Low ODs or false-negative results
1. Pipette function.
2. Not freshly coated plates.
3. Incomplete reagent mixing.
4. Short incubation.
5. Low temperature.
6. Incorrect plate reader ﬁlters.
7. Wrong buffers.
G Increased CVs for duplicates.
1. Poor washing.
2. Alignment issues causing carryover.
G No color development.
1. Incorrectly diluted or made conjugate.
2. Wrong order of reagent additions.
3. Wrong reagents or stop solution used as reagent.
4. Missed or mistaken pipetting.
5. Poor repeatability: technical accuracy, pipet-
ting too fast, carryover between wells, inconsis-
tent reagents, time variability, temperature
variability, contamination, wrong ﬁlter, sample
The ELISA method provides an ideal tool for a wide range
of studies in many biological and medical laboratories.
The method has high sample handling capacity, reliable
analytical performance, and ease of use. It can be manually
performed or run on automatic or semiautomatic plat-
forms. ELISA technology depends on complexity as well
as ﬂexibility in the reagents rather than in instrumentation
alone, therefore, the design of the assay is very important.
However, the technical simplicity of ELISA belies the
fairly complex course of ELISA development. Selecting
key raw materials, optimizing the reagent formulation, and
deﬁning the processes for consistently making reagents are
critical factors for developing ELISA methods. At each
step, there are considerations to be made, trade-offs to
consider, a balance to be achieved. And it is essential to
ﬁnd this balance within each step, as well as across the
entire design process as a whole. It is also important to
keep in mind that there is no “correct” combination of
materials and conditions; the right balance depends on the
speciﬁc needs that have been deﬁned for the assay system.
Ultimately, to view the development process as an inter-
related system rather than many individual factors helps
establish optimal conditions for assay. From this perspec-
tive, the system balance can be adjusted as a whole, giving
that there is no absolute, only relative.
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